Cell adhesion to protein blots on nitrocellulose ("NC") following fractionation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was first published in 1982 (Hayman, E. G., Engvall, E., A'Hearn, E., Barnes, D., Pierschbacher, M., and Ruoslahti, E., J. Cell Biol. 95:20-23 (1982)). Methods for staining and detection of cells adherent to NC were described in 1986 (Seshi, B., Anal. Biochem. 157:331-342 (1986)). However, despite the apparent simplicity in concept, the cell blotting technique has been used only on occasion (Wong, H. J., Aubin, J. E., Wasi, S., and Sodek, J., Biochem. J. 232: 119-123 (1985); Ward, H. D., Lev, B. I., Kane, A. V., Keusch, G. T., and Pereira, M. E. A., Biochemistry 26: 8669-8675 (1987); Campbell, A. D., Long, M. W., and Wicha, M. S., Nature 329: 744-746 (1987); Ferro, V., D'Arrigo, C., Ogden, D., and Evans, C. W., Biochem. Soc. Transactions 16: 144-146 (1988); Isberg, R. R., and Leong, J. M., Proc. Natl. Acad. Sci. USA 85: 6682-6686 (1988)) and has not become a mainstream technique of biological investigation like the southern (Southern, E. M., J. Mol. Biol. 98:503-517 (1975)) and western blotting techniques, (Towbin, H. Staehelin, T., and Gordon, J., Proc. Natl. Acad. Sci. USA 76:4350-4354 (1979); Burnette, W. N., Anal. Biochem. 112:195-203 (1981)). There are two potential reasons why the technique has failed to achieve practical application. Firstly, prior to a successful cell adhesion assay on blots, cell adhesion molecules ("CAM") need to be extracted, fractionated, and blotted in a functional form; i.e., they need to withstand exposure to various detergents, as well as other steps in electrophoresis and blotting procedures, while still retaining their ability to mediate physiologically relevant adhesion function. This question has not been systematically studied in relation to the CAMs. Secondly, NC paper is not the best membrane available for protein blotting. It is fragile, not very resistant to chemicals, and difficult to handle during the cell adhesion assay. NC also does not retain protein well, thus potentially compromising sensitivity. However, practical methods for detecting cells adherent on PVDF membranes have not been described.
It has been hypothesized that normal hematopoiesis occurs as a consequence of complex interactions of hematopoietic progenitor cells ("HPC") with proteins expressed on marrow stromal cell/fibroblast membranes, as well as extracellular matrix proteins and soluble mediators secreted by these cells (Torok-Storb B., Blood 72:373 (1988)). Various investigators have shown that normal HPC and myeloid leukemic blast cells alike exhibit interaction and adhesion to cultured bone marrow ("BM") stromal cells/fibroblasts (Liesveld J. L., Abboud C. N., Duerst R. E. , Ryan D. H., Brennan J. K., Litchman M. A., Blood 73: 1794 (1989); Liesveld J. L., Winslow J. M., Kempski M. C., Ryan D. H., Brennan J. K., Abboud C. N., Exp. Hematol. 19: 63 (1991); Gordon M. Y., Dowding C. R., Riley G. P., Goldman J. M., Greaves M. F., Nature 328:342 (1987)). Of special significance is the finding that HPC and leukemic blasts not only exhibit adherence to cultured marrow stromal cells/fibroblasts but actually need them to escape from apoptosis and for proliferation in culture (Manabe A., Coustan-Smith E., Behm F. G., Raimondi S. C., Campana D., Blood 79:2370 (1992)). Stromal cells and fibroblasts represent populations of supporting cells of the BM microenvironment. In practice, fibroblasts are grown by culturing BM in a standard culture medium containing fetal bovine serum (FBS), whereas a stromal cell layer is grown by supplementing the standard cell culture medium-with horse serum and hydrocortisone (Dexter-type cultures). Stromal cells are morphologically heterogeneous and exhibit adipocyte differentiation whereas fibroblasts do not show such features. Stromal cells play an important supportive role in myelopoiesis whereas fibroblasts are required for in vitro lymphopoiesis. Therefore, it is of great interest to investigate interactions of HPC and their leukemic counterparts with the stromal cells as well as fibroblasts.
In vitro proliferation of normal human and murine lymphoid progenitors requires adhesion to BM fibroblasts mediated by the integrin adhesion protein VLA-4 ("very late antigen-4 or .alpha.4/.beta.1 integrin or CDw49d/CD29") (Kincade P. W., Sem. Immunol. 3:379 (1991)). The role of known adhesion molecules in myelopoiesis and proliferation of leukemic cells is less clear. Tavassoli et al have shown that a 110 Kd membrane protein on murine myeloid HPC mediates their adhesion to stromal cells and, in fact, inhibition of this adhesion blocks growth of the HPC (Tavassoli M., Aizawa S., Matsuoka T., Hardy C., in Golde D. W. (ed), Hematopoiesis, New York, N.Y., Alan R. Liss, Inc. (1990), p 145; Aizawa S., Tavassoli M., Proc. Natl. Acad. Sci. USA 84:4485 (1987)). A corresponding ligand protein of 37 Kd has recently been identified on murine BM stromal cells (Shiota Y., Wilson J. G., Harjes K., Zanjani E. D., Tavassoli M., Blood 82: 1436 (1993)). Therefore, adhesion to marrow stromal cells appears to play a pivotal role at least in normal hematopoiesis. However, no human counterpart involved in homing of HPC to marrow has been identified. Some reports suggest that VLA-4 and its ligand VCAM-1 (vascular cell adhesion molecule-1) are partially responsible for adherence between myeloid HPC and BM stromal cells (Miyake K., Hasunuma Y., Yagita H., Kimoto M., J. Cell Biol. 119: 653 (1992); Simmons P. J., Masinovsky B., Longenecker B. M., Berenson R., Torok-Storb B., Gallatin W. M., Blood 80: 388 (1992)). Based on failure to abrogate complete binding between HPC and BM stroma using anti-VCAM-1 antibodies alone or in combination with EILDVPST (Seq. I.D. No. 1) peptide, the latter investigators concluded a probable role for other unknown CAMs in mediating adhesion between HPC and BM stroma (Simmons P. J., Masinovsky B., Longenecker B. M., Berenson R., Torok-Storb B., Gallatin W. M., Blood 80:388 (1992)).
The conventional method for investigating unknown CAMs is indirect and requires generating MAbs to whole cells or plasma membrane components and then using the MAbs to block cell-cell adhesion (Bevilacqua M. P., Pober J. S., Mendrick D. L., Cotran R. S., Gimbrone Jr M. A., Proc. Natl. Acad. Sci. USA 84: 9238 (1987)). However, use of whole cells or crude mixtures of proteins as immunogens may present the problem of antigenic competition, the situation in which one antigen can suppress the response to another and consequently antibodies may not form for antigens of interest (Thaihamer J., Freund J., J. Immunol. Methods 80: 7 (1985); Hammerl P., Weger R., Thaihamer J., Mol. Immunol. 25: 313 (1988)). Even if MAbs are developed, they may not have the functional blocking activity. Even if they have the blocking activity, they may not identify the molecule of interest at the biochemical level for the following reason. The conventional technique that has been used for biochemical detection of some of the known CAMs such as ELAM-1 (endothelial-leukocyte adhesion molecule-1) (Bevilacqua M. P., Pober J. S., Mendrick D. L., Cotran R. S., Gimbrone Jr. M. A., Proc. Natl. Acad. Sci. USA 84: 9238 (1987)); CD54 (Myers C. L., Desai S. N., Schembri-King J., Letts G. L., Wallace R. W., Am. J. Physiol. 262:C365 (1992)); ICAM-2 (intercelluglar adhesion molecule-2) (de Fougerolles A. R., Stacker S. A., Schwarting R., Springer T. A., J. Exp. Med. 174:253 (1991)); VCAM-1 (Simmons P. J., Masinovsky B., Longenecker B. M., Berenson R., Torok-Storb B., Gallatin W. M., Blood 80: 388 (1992)); VLA-4 (Holzmann B. , Mcintyre B. W., Weissman I. L., Cell 56:37 (1989)); CD44 (Stamenkovic I., Amiot M., Pesando J. M., Seed B., Cell 56:1057 (1989)); and CD61 (Rossi G. D., Zarcone D., Mauro F., Cerruti G., Tenca C., Puccetti A., Mandelli F., Grossi C. E., Blood 81: 2679 (1993)) consists of, a) Labeling of cell surface proteins, b) Extraction of the labeled molecules by NP-40 (nonidet P-40) or Triton.RTM. X-100 ranging in concentration from 0.5% to 2%, c) Immunoprecipatation of the solubilized molecules using an appropriate MAb (Kessler S. W., in Langone J. J., Vunakis H. V. (eds), "Methods in Enzymology," vol. 73, New York, N.Y., Academic Press (1981) p. 442), and d) SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography or western blotting if the cells had been biotin-labeled instead of radiolabeled. While most known CAMs are extractable by Triton.RTM. X-100 or NP-40 and, therefore, can be detected by the standard immunoprecipatation technique, it is apparent that the standard technique would not identify new CAMs if they are not solubilized by Triton.RTM. X-100 or NP-40.